Membrane Trafficking of Renal Potassium Channel

描述（由申请人提供）：ROMK钾（K+）通道存在于肾皮质集管（CCD）主要细胞的顶膜中，负责肾单元段腔内K+的分泌。CCD中K+的分泌受膳食K+摄入量和利尿激素的调节。研究表明，饮食中K+的摄入和抗利尿激素通过改变CCD中活性K+通道的数量，至少部分地改变了K+的分泌。这是如何发生的尚不完全清楚，但可能涉及到通道的膜运输的改变和/或沉默通道的激活。我们的长期目标是了解肾K+通道膜运输的分子机制及其在通道生理调节中的重要性。胞吞作用有多种途径。我们的初步结果表明，ROMK的内吞作用是通过网格蛋白包被囊泡（CCVs）介导的。申请的第一部分的具体目的是检查参与通道内吞作用的ROMK和ccv的结构元件，并检查低饮食K+摄入量是否通过增加内吞作用和随后的ROMK降解来减少K+通道。ROMK与ccv的关联将通过免疫荧光共定位、生化纯化和免疫共沉淀来研究。利用双电极电压钳和膜片钳记录技术研究非洲爪蟾卵母细胞和CCD中蛋白相互作用的功能意义。我们还发现CCD顶端膜上的一些ROMK通道受到syntaxin-lA的抑制。应用程序的第二部分是测试抗利尿激素通过激活预先存在的沉默通道来增加活跃通道密度的假设。将进行生化结合试验和膜片钳记录来检验这一假设。

DESCRIPTION (provided by applicant): ROMK potassium (K+) channels are present in the apical membrane of principal cells of the kidney cortical collecting ducts (CCD) and are responsible for luminal K+ secretion in this nephron segment. K+ secretion in CCD is regulated by dietary K+ intake and the hormone vasopressin. It has been shown that dietary K+ intake and vasopressin alters K+ secretion, at least partly, by altering the number of active K+ channels in CCD. How this occurs is not fully understood, but may involve alteration of membrane trafficking of the channels and/or activation of silent channels. Our long-term objectives are to understand the molecular mechanism for membrane trafficking of renal K+ channels and its importance in physiological regulation of the channels. There are multiple pathways for endocytosis. Our preliminary results indicate that endocytosis of ROMK is mediated via clathrin-coated vesicles (CCVs). The specific aims of the first part of the application are to examine the structural elements of ROMK and CCVs involved in the endocytosis of the channels and to examine whether low dietary K+ intake decreases K+ channels by increasing endocytosis and subsequent degradation of ROMK. Association of ROMK with CCVs will be studied by immunofluorescent colocalization, biochemical purification, and immunocoprecipitation. Functional significance of protein interaction will be examined using two-electrode voltage clamp and patch-clamp recording in Xenopus oocytes as well as CCD. We also found that some of the ROMK channels in the apical membrane of CCD are inhibited by syntaxin-lA. The second part of the application is to test the hypothesis that vasopressin increases density of active channels by activating pre-existing silent channels. Biochemical binding assay and patch-clamp recording will be performed to examine this hypothesis.